Although flagella, motility and/or chemotaxis have been implicated as important for biofilm formation in other organisms (Lawrence et al., 1987; Korber et al., 1989; 1994; DeFlaun et al., 1994; Graf et al., 1994; Mills and Powelson, 1996; Vidal et al., 1998), their precise roles in this process have not yet been defined. We reasoned that there are three mechanisms through which flagella might be required. First, it is possible that flagella could be directly required for attachment to abiotic surfaces, thus facilitating the initiation of biofilm formation (e.g. as with tethered cells). Alternatively, motility could be necessary to enable a bacterium to reach the surface (e.g. to move through surface repulsion present at the medium-surface interface). Also, motility might be required for the bacteria within a developing biofilm to move along the surface, thereby facilitating growth and spread of the biofilm. Finally, it is possible that chemotaxis is required for the bacteria to swim towards nutrients associated with a surface.

As flagellar synthesis, motility and chemotaxis have been extensively studied in E. coli (Macnab, 1996; Stock and Surette, 1996), well-defined mutations that inhibit each of these three aspects of flagellar function are available. Accordingly, we obtained the following mutations: (i) fliC ::kan (strains harbouring this allele are unable to synthesize flagellin) and flhD ::kan (a master regulator of flagellar synthesis whose absence confers an inability to synthesize flagella); (ii) motA, motB and motAB (lesions that do not inhibit flagellar biosynthesis but render cells non-motile or paralysed); and (iii) cheA-Z ::kan (strains harbouring this lesion are motile but non-chemotactic).

Each of these alleles was moved into 2K1056 via P1vir transduction, and the resulting strains were analysed for their ability to form biofilms. Construction of these strains provided us with the tools required to distinguish between the possible roles of flagella/motility/chemotaxis that were detailed above. First, the simple microtitre dish assay used in our screen revealed that motile cells that are non-chemotactic ( cheA-Z ::kan) appear to form biofilms indistinguishable from their wild-type counterpart. In contrast, cells either lacking flagella (fliC ::kan, flhD ::kan) or possessing paralysed flagella ( motA, motB, or motAB ) were severely defective in biofilm formation (Fig. 2). When biofilm formation was quantified over time (Experimental procedures), it became very clear that, under these conditions, chemotaxis is completely dispensable for normal biofilm formation (Fig. 3). In contrast, cells either lacking complete flagella (fliC ::kan) or possessing paralysed flagella ( motA, motB, or motAB ) are severely hindered in the initial stages of biofilm formation (Fig. 3).

More detailed analysis of the defects conferred by these alleles was obtained through microscopic analysis of cells attached (or the absence of such attached cells) to PVC after growth in LB. As illustrated in Fig. 4A and B, motile cells that are non-chemotactic are able to form biofilms that are indistinguishable at the cellular level from the biofilms formed by wild-type cells. In contrast, non-flagellated or paralysed cells attach poorly to PVC. Moreover, the few cells that do attach are often located in small, dense clusters of cells (Fig. 4D).

Type I pili are critical for initial attachment to abiotic surfaces

As mentioned above, the macroscopic analysis of biofilm formation of fim mutants was analogous to that observed with the motility-defective mutants (i.e. clear wells after staining with CV) (Fig. 2). However, microscopic analysis of these mutants revealed distinct phenotypes. Specifically, fim mutants are even more dramatically defective in initial attachment than are the paralysed and non-flagellated cells. As illustrated in Fig. 4C, most microscopic fields had no cells attached at all, and only infrequently were a few attached cells observed. This observation indicated that type I pili are critical for initial interaction with abiotic surfaces such as PVC.